Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/67—Vitamins
  • A61K8/676—Ascorbic acid, i.e. vitamin C
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
  • A61K8/0241—Containing particulates characterized by their shape and/or structure
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/16—Emollients or protectives, e.g. against radiation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/02—Nutrients, e.g. vitamins, minerals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
  • A61Q19/08—Anti-ageing preparations
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
  • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
  • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
  • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D307/62—Three oxygen atoms, e.g. ascorbic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
  • A61K2800/41—Particular ingredients further characterized by their size
  • A61K2800/413—Nanosized, i.e. having sizes below 100 nm
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
  • A61K2800/60—Particulates further characterized by their structure or composition
  • A61K2800/65—Characterized by the composition of the particulate/core
  • A61K2800/652—The particulate/core comprising organic material
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T428/00—Stock material or miscellaneous articles
  • Y10T428/29—Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
  • Y10T428/2982—Particulate matter [e.g., sphere, flake, etc.]

Definitions

• the present invention aims to propose a new bioconjugate of vitamin C particularly interesting for its activity with respect to the skin.
• the present invention also relates to processes for its preparation, compositions comprising it and its use in the cosmetic, dermatological, pharmaceutical and food fields.
• Human skin consists of two compartments namely a superficial compartment, the epidermis, and a deep compartment, the dermis, which provides the epidermis with its solid support.
• This dermis nourishing tissue of the epidermis, consists mainly of fibroblasts and an extracellular matrix composed mainly of collagen, elastin and a substance called the fundamental substance, components synthesized by the fibroblast.
• the fibroblasts mainly synthesize collagens, matrix glycoproteins other than collagen (fibronectin, laminin), proteoglycans and elastin.
• the keratinocytes mainly synthesize sulfated GAGs and hyaluronic acid.
• the extracellular matrix of the dermis As for the extracellular matrix of the dermis, it is like that of all the connective tissues of the body, composed of proteins belonging to several large families: collagens, matrix glycoproteins other than collagens (fibronectin, laminin), elastin and proteoglycans (PGs). Glycosaminoglycans (GAGs) are also found in free form (ie not bound to a protein).
• the components of the extracellular matrix play an important role in the mechanical properties of the skin, in particular in its tone, firmness, elasticity and flexibility.
• Collagen is a protein composed of three polypeptide alpha chains linked by hydrogen bonds between hydroxy lysine and hydroxyproline and associated covalent bonds. According to the various possible combinations, there are several types of collagen with structures of their own, found in particular organs. Among these types of collagen, there may be mentioned type I collagen and type III collagen which are located mainly within the dermis. Type I collagen plays a role in stiffness and tissue resistance. During aging, it is more and more predominant (more than 80%). Thus, the fibers become stiffer and less flexible, resulting in a loss of firmness of the skin.
• type III collagen also called “youth collagen”
• young collagen it is synthesized in abundance during fetal life and adolescence.
• vitamin C also known as ascorbic acid, of 5- (1, 2-dihydroxyethyl) -3,4-dihydroxy-5H-furan-2-one or 3 - keto-L-gulofuranolactone, is involved in hundreds of processes in the body.
• Vitamin C is also known to help the body make collagen, which is essential for the formation of connective tissue in the skin, ligaments and bones.
• an organism is supplemented with vitamin C orally, typically via food and, more occasionally, via dietary supplements enriched with vitamin C. More recently, it has been proposed to administer topically vitamin C, in particular for the purpose of improving the aesthetic appearance of the skin.
• vitamin C is also known to be one of the most fragile vitamins. Thus, it is sensitive to heat, fears basic media and above all is very sensitive to oxygen in the air.
• vitamin C as such has a low penetration into the skin due in particular to its hydrophilic nature.
• liposoluble derivatives comprising vitamin C in a form coupled at its 6-position to a polyunsaturated fatty acid (6-O-PUFA).
• Other vitamin C derivatives such as the C2-C20 aliphatic carboxylic acid esters at the 6-position of ascorbic acid such as 6-ascorbyl palmitate and 6-ascorbyl linolate are also described in US Pat. No. 4,997,958 but for purposes of antioxidants.
• the present invention specifically aims to provide new vitamin C derivatives giving satisfaction in these terms.
• the present invention aims to provide new vitamin C derivatives which penetrate more easily into the skin and which improve the beneficial effect of vitamin C in the deep layers of the skin.
• the present invention aims at providing novel vitamin C derivatives capable of exhibiting an increased stability with regard to oxidation.
• the present invention aims at providing new vitamin C derivatives capable of stimulating the expression of collagen, and more particularly of collagen III.
• the present invention aims to provide new vitamin C derivatives capable of stimulating the expression of GAGs.
• the present invention aims to propose novel vitamin C derivatives, which are suitable for formulation both in a medium aqueous in an oily medium, able to be formulated homogeneously, even for significant amounts of vitamin C equivalent.
• the present invention aims at providing new vitamin C derivatives compatible with any mode of administration and giving satisfaction in terms of safety.
• the present invention aims to provide new vitamin C derivatives capable of improving the general morphology of the skin in terms of flexibility, hydration, thickness, elasticity.
• an object of the present invention relates to a complex formed of at least one molecule of 5- (1,2-dihydroxy-ethyl) -3,4-dihydroxy-5H-furan-2-one or covalently linked derivative.
• - mi 1, 2, 3, 4, 5 or 6;
• m 2 0, 1, 2, 3, 4, 5 or 6; and represents the linkage to the 5- (1,2-dihydroxy-ethyl) -3,4-dihydroxy-5H-furan-2-one or derivative molecule.
• the term complex or conjugate will be used interchangeably to designate the coupling product between at least one molecule of 5- (1, 2-dihydroxy-ethyl) -3,4-dihydroxy-5H-furan-2 -one or derivative and at least one hydrocarbon radical of formula (A).
• connection associated with the symbol means that the binding site of the entity of formula (A) to the ascorbic acid or derivative entity.
• the binding site may be involved either directly in a simple covalent bond between the entity (A) and the ascorbic acid entity or derivative, or be represented by a function resulting from the interaction between two reactive functions, for example between a carboxylic function and an alcohol function, or be represented by a group of spacer type.
• the entity (A) and the ascorbic acid entity or derivative may be bonded to each other via a covalent bond or via a spacer group or an ester, ether, phosphate or amide functional group, such as explained below.
• the invention is a complex or conjugate as defined above, wherein the compound or hydrocarbon radical comprises from 12 to 40 carbon atoms, preferably from 18 to 30 carbon atoms.
• such a compound or hydrocarbon radical may advantageously be derived from squalene, farnesol or geraniol and more preferably from squalene.
• the two types of entities forming the complex defined above are coupled by a covalent bond of the ester, ether, phosphate or amide type, and preferably an ester.
• the present invention also relates to an oily formulation comprising a complex or conjugate as described above. More particularly, this formulation is in the form of a gel which can contain up to at least 3% by weight, or even at least 10% by weight, expressed as weight of vitamin C active, relative to the total weight of the composition.
• the oil considered for formulating the complex according to the invention in the state of such a gel is squalene or one of its derivatives.
• the present invention relates to nanoparticles of a complex or conjugate as described above.
• the conjugate according to the invention is capable of self-assembly, that is to say spontaneously self-organize in an aqueous medium in the form of nanoparticles which have at least the same activities as the conjugate itself.
• the present invention also relates to a method for preparing said nanoparticles comprising at least the dispersion of a complex according to the present invention, in at least one organic solvent, at a sufficient concentration. to obtain, during the addition of the corresponding mixture, with stirring, an aqueous phase, the instantaneous formation of nanoparticles in suspension in said aqueous phase, and, where appropriate, the isolation of said nanoparticles.
• the nanoparticulate state of the vitamin C complex according to the invention advantageously makes it possible to formulate it so as to be uniformly stabilized in an aqueous medium.
• the nanoparticulate state of the vitamin C complex according to the invention makes it possible to increase the penetration thereof to the deep layers of the skin.
• This improvement in the penetration into the deep layers of the skin advantageously makes it possible to use lower concentrations of vitamin C than those usually required.
• nanoparticles according to the invention advantageously make it possible to stimulate the expression of collagen and more specifically of collagen III, as well as GAGs, as indicated previously, and to improve the general morphology of the skin.
• this particular form is administrable in the form of an aqueous suspension parenterally (or injectable) and especially intravenously.
• the average size of these nanoparticles varies from 30 nm to 500 nm, in particular from 40 to 250 nm, or even from 45 to 95 nm.
• Nanoparticles of conjugates formed by coupling at least one hydrocarbon radical and, in particular squalene, with an active agent such as gemcitabine, nucleosides, nucleic acids, statins, taxoids, doxorubicin, epirubicin and beta-lactam have already been described in WO 2006/090029, Couvreur et al, Nano Lett. 2006, 6, pp. 2544-25 48, WO 2009/150344, WO 2009/071850, WO 2010/049899 and WO2010 / 049900.
• the present invention also relates to a cosmetic, dermatological or food composition
• a cosmetic, dermatological or food composition comprising as active ingredient at least one complex according to the invention and / or nanoparticles according to the invention, in combination with at least one physiologically acceptable vehicle.
• the invention relates to the cosmetic use of a complex according to the invention and / or nanoparticles according to the invention for the prevention and / or treatment of signs of aging of the skin of the body and / or of the face.
• a cosmetic composition according to the invention can be used for the prevention and / or treatment of wrinkles and / or fine lines, wilted skin, lack of elasticity and / or tone of the skin, of the skin. thinning of the dermis, degradation of coUagene fibers, soft skin, thinned skin and / or internal skin damage from exposure to ultraviolet radiation.
• the present invention aims at protecting the cosmetic use of a complex or conjugate according to the invention and / or nanoparticles according to the invention for stimulating the synthesis of coUagene, in particular coUagene III, and / or stimulating the synthesis of glycosaminoglycans.
• the present invention also relates to a complex or conjugate according to the invention and / or nanoparticles according to the invention for its use in medicine and more specifically in the prevention and / or treatment of burns and / or wounds and / or any another pathology related to scarring of the dermis and / or epidemic.
• the present invention relates to a cosmetic treatment method for the skin of the body and / or face and / or scalp, in which a composition as defined above is administered.
• the complexes according to the invention are formed by coupling a molecule of vitamin C or the like to at least one specific hydrocarbon-based radical.
• Radical hydrocarbon a molecule of vitamin C or the like to at least one specific hydrocarbon-based radical.
• hydrocarbon radical corresponds to formula (A) as follows:
• - mi 1, 2, 3, 4, 5 or 6;
• m 2 0, 1, 2, 3, 4, 5 or 6; and represents the molecule binding site the 5- (1,2-dihydroxyethyl) -3,4-dihydroxy-5H-furan-2-one molecule or derivative.
• This hydrocarbon radical generally derives from the placing in contact with at least one comose molecule of formula ( ⁇ '):
• Y represents a reactive function and in particular of the alcohol, carboxylic acid or phosphate type
• mi and m 2 are as defined for the radical of formula (A).
• the hydrocarbon compound of formula ( ⁇ ') comprises at least 12 carbon atoms, in particular from 12 to 40 carbon atoms and preferably from 18 to 30 carbon atoms.
• the compounds of formula ( ⁇ ') are chosen from compounds of formula ( ⁇ ') in which:
• - mi and m 2 represent 0 and Y is a -COOH function
• a compound of formula (A) useful for the formation of a conjugate according to the present invention is squalene (also called spiracene or sirprene), which is an essential intermediate for the biosynthesis of cholesterol.
• farnesol which is an acyclic sesquiterpene alcohol.
• Yet another useful compound for the formation of a conjugate according to the present invention is geraniol (also called rhodinol) which is an unsaturated terpene alcohol.
• the hydrocarbon derivative present in a complex according to the present invention is a squalene derivative.
• hydrocarbon compounds capable of forming a complex containing at least one squalene derivative according to the present invention mention may be made more particularly of squalene acid and its derivatives such as 1, 2-trisorsqualene acid, 1 , 2-tris-norsqualenamine, 1, 2-tris-norsqualenol, 1,2-tris-norsqualethiol, squalenacetic acid, squalenylethanol, squalenylethanethiol, squalenylethylamine.
• squalene acid and its derivatives such as 1, 2-trisorsqualene acid, 1 , 2-tris-norsqualenamine, 1, 2-tris-norsqualenol, 1,2-tris-norsqualethiol, squalenacetic acid, squalenylethanol, squalenylethanethiol, squalenylethylamine.
• a conjugate according to the present invention may contain at least one radical derived from the covalent coupling of a 1, 2-troinsortene acid molecule.
• a conjugate according to the present invention may contain at least one radical derived from the covalent coupling of a 1, 2-trisorsqualenol molecule.
• a conjugate according to the present invention may comprise at least two hydrocarbon radicals according to the present invention.
• vitamin C and its derivatives within the meaning of the invention have in common a 3,4-dihydroxy-5H-furan-2-one system that can be schematized by the pattern as follows.
• vitamin C is represented by formula (IV) and its tautomeric form as follows.
• the compounds of general formula (IV) may comprise one or more asymmetric carbons. They can therefore exist as enantiomers or diastereoisomers. These enantiomers, diastereoisomers, as well as their mixtures, including the racemic mixtures, form part of the invention.
• the compounds of the aforementioned formulas may exist in the form of acids or of addition salts with bases.
• salts are advantageously prepared with pharmaceutically acceptable bases, but the salts of other bases which are useful, for example, for the purification or separation of the compounds of the abovementioned formulas also form part of the invention.
• the compounds of general formula (IV) may, in addition, be in the form of hydrates or solvates, namely in the form of associations or combinations with one or more molecules of water or with a solvent. Such hydrates and solvates are also part of the invention.
• ascorbic acid sodium ascorbate, potassium ascorbate and / or calcium ascorbate, and preferably ascorbic acid.
• the conjugate according to the invention is of general formula (I)
• R 1, R 2 , R 3 and R 4 which are identical or different, represent, independently of one another, a hydrogen atom or a hydrocarbon radical of formula (A) as defined above,
• R 1, R 3 and R 4 are as defined above, at least one of the groups R 1, R 3 and R 4 being different from the hydrogen atom.
• the conjugate according to the invention is of formula
• R 2 , R 3 and R 4 are as defined above and R '1 represents a hydrocarbon radical of formula (A) as defined above.
• the complex or conjugate according to the invention is of general formula (IA) in which R 2 , R 3 and R 4 all represent a hydrogen atom.
• the hydrocarbon radicals according to the invention are generally bound by a covalent bond at the level of the hydroxyl group carried by the carbon atom 2 and / or 3 and / or 4 and / or 5 and / or 6 of the ascorbic acid, and preferably at group level hydroxyl borne by the carbon atom 6 of ascorbic acid, or at the hydroxyl group borne by the carbon atom 2 of ascorbic acid, or at the level of the hydroxyl group borne by the carbon atom 3 of ascorbic acid.
• the ascorbic acid complexes according to the present invention may be complexes comprising two derivatizations, three derivatizations or even four derivatizations, these may be identical or different.
• the formation of the vitamin C complex / hydrocarbon radical according to the invention requires that the two entities put together respectively carry a so-called reactive function, that is to say capable of forming via their interaction the covalent bond expected. These functions may or may not be naturally present on the two starting entities. In the negative, the starting entity will have to undergo a modification, prior to the coupling reaction.
• the hydrocarbon compound according to the invention is generally carrying a function capable of reacting with a function present on the molecule of vitamin C considered, so as to establish a co-valid bond between the two entities, for example of ester type , ether, phosphate or amide, thereby forming a covalent complex.
• the hydrocarbon compound capable of reacting with a molecule of vitamin C or one of its derivatives to form the aforementioned complex is 1, 1 ', 2-tris-norsqualenic acid or one of its derivatives as for example its acid halide and more particularly the acid chloride, or its mixed anhydride with ethyl chloroformate.
• the acid chloride derived from 1,1 ', 2-tris-norsqualenic acid is used.
• the hydrocarbon compound capable of reacting with a molecule of vitamin C or one of its derivatives to form the abovementioned conjugate is 1,1 ', 2-tris-norsqualenol or one of its derivatives, for example 1,1 ', 2-tris-norsqualenol mesylate.
• the covalent link existing between the two types of molecules can be represented by a functional spacer or linker.
• a functional spacer or linker may especially be useful for increasing the strength of the vitamin C / hydrocarbon radical interaction.
• Such an arm may also be advantageous for introducing, via each of the two ends of its skeleton, the appropriate functions, that is to say respectively possessing the expected reaction affinity, one for the function present on the compound with a hydrocarbon structure according to the invention, and the other for the function present on the vitamin C molecule considered.
• this linker also has at its skeleton a labile function, which is suitable for the separation of the hydrocarbon structure compound from the vitamin C molecule under consideration. It may for example be a peptide motif recognizable by an enzyme.
• linking arms that can be envisaged according to the invention, mention may be made in particular of the alkylene chains, the (poly) amino acid, polyol, saccharide and polyethylene glycol (polyetheroxidic) units.
• Alkylene chains means a C 1 -C 4 alkylene group, that is to say a divalent alkyl group which may comprise from 1 to 4 carbon atoms.
• alkylene chains means a C 1 -C 4 alkylene group, that is to say a divalent alkyl group which may comprise from 1 to 4 carbon atoms.
• saccharide unit a radical comprising at least one radical chosen from trioses (glyceraldehyde, dihydroxyacetone), tetroses (erythrose, threose, erythrulose), pentoses (arabinose, lyxose, ribose, deoxyribose, xylose, ribulose, xylulose) , hexoses (allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose, tagatose), heptoses (mannoheptulose, sedoheptulose), octose (octolose, 2-keto-3-deoxy-manno-octonate) ), isonoses (sialosis), and
• (poly) amino acid motif means a unit having at least one unit:
• n is greater than or equal to 1 and R 'represents a hydrogen atom, a C 1-6 alkyl group, optionally substituted with one or more hydroxyls, a C 1-6 alkoxy group.
• a "covalent bond” preferably represents a covalent bond especially as specified above, but also covers a covalent bond represented by a linker as defined above.
• This reaction is generally carried out in solution in the presence and in excess of at least one hydrocarbon compound considered according to the present invention with respect to the vitamin C molecule used according to the invention, for example at the rate of two equivalents, according to the standard conditions required to interact the two specific functions carried by each of the two entities.
• a hydrocarbon compound starting for the synthesis of a complex according to the invention is a squalene derivative in acid form, such as, for example, 1, 1 ', 2-tris-norsqualenic acid, which can be prepared according to method described in Example la.
• the present invention relates to a process for the preparation (hereinafter referred to as the first route) of the complex according to the invention comprising:
• the first preparation route of the complex according to the invention makes it possible to obtain amounts of said complex with a yield of at least 30%, or even at least 34%, and with a degree of purity of approximately 90%.
• the invention relates to a process for the preparation (hereinafter referred to as the second route) of the conjugate according to the invention comprising the esterification reaction between at least one molecule of 5- (1,2-dihydroxyethyl) -3,4-dihydroxy-5H-furan-2-one of formula (IV)
• said esterification reaction is carried out in the presence of a lipase as a catalyst.
• Lipases or triacylglycerol acyl hydrolases are atypical enzymes by their mechanism of action and substrate specificity (Fickers et al., Biotechnol, Agron Soc., 2008, 12 (2), 119-130 or Alloue et al. Biotechnol, Agron, Soc. 2008, 12 (1), 57-68). They are catalysts very widely used in organic synthesis, mainly because of their stability and their activity in a solvent medium.
• the lipases in accordance with the invention there may be mentioned in particular plant lipases, mammalian lipases and microbial lipases.
• a microbial lipase even more preferentially, a lipase chosen from C. cylindracea, C. antarctica, C. miehei and mixtures thereof, and even more preferably a C. antarctica lipase such as that marketed. by the company Novozyme Corp under the trade name Novozyme 435 ® .
• the second route of preparation of the complex according to the invention leads to obtaining amounts of said complex with a yield less than or equal to 30% and with a degree of purity greater than or equal to 95%.
• the present invention relates to a process for preparing the conjugate according to the invention comprising:
• R, R 'and R represent a hydrogen atom or a protecting group
• mi and m 2 are as defined above for the compound of formula (A); and Z is a hydrogen atom or a group -S0 2 -CH 3 .
• the hydrocarbon-based starting compound for the synthesis of the conjugate according to the invention is 1,1 ', 2-trisnorsqualenol as illustrated in example lb.
• the covalent coupling of the two entities of the conjugate according to the invention can be carried out in particular as follows.
• a process for the preparation of the conjugate according to the invention comprising the reaction of at least one alcohol molecule of formula V)
• the hydrocarbon compound starting for the synthesis of the conjugate according to the invention is the 1,1 ', 2-trisnorsqualenol mesylate as illustrated in particular in Example 1c.
• a process for the preparation of the conjugate according to the invention comprising the reaction of at least one alcohol monoxide molecule of formula (VII)
• the subject of the present invention is also a complex according to the invention formulated in the gel state with at least one oil.
• other oils can be used to the extent that they are compatible with the intended applications and the complex according to the invention.
• a gel formed from squalene and a complex or conjugate according to the invention makes it possible in particular to obtain homogeneous formulations which can comprise up to at least 3% by weight, or even at least 10% by weight, expressed by weight of d vitamin C active relative to the total weight of the composition.
• Such a gel is more particularly advantageous for topical administration.
• It may also be in the form of cream, ointment, ointment or any other type of formulation known to those skilled in the art and suitable for the intended applications.
• the inventors have found that the complex formed by covalent coupling of at least one vitamin C molecule considered according to the invention with at least one hydrocarbon compound within the meaning of the invention shows an ability to organize under a compacted form in a polar solvent medium, and thus leads to the formation of nanoparticles.
• the present invention is directed to nanoparticles of the complex according to the invention.
• the nanoparticles thus obtained have an average size ranging from 30 nm to 500 nm, in particular from 40 nm to 250 nm, or even from 45 nm to 95 nm, measured by light scattering using the nanosizer.
• these particles have a size that is compatible with any mode of administration, in particular topical, injectable and oral.
• the nanoparticulate formulation is advantageous insofar as it makes it possible in particular to formulate in a stabilized manner the complex according to the invention in an aqueous medium and to increase the penetration of vitamin C to the deep layers of the skin.
• the nanoparticles according to the invention are, of course, capable of bringing to the surface a multitude of reactive functions, such as hydroxyl or amine functions for example. It is therefore conceivable to fix these functions all kinds of molecules, including covalent bonds.
• marker-type molecules compounds capable of providing a targeting function, as well as any compound capable of conferring on them characteristics. specific pharmacokinetics.
• lipophilic derivatives of polyethylene glycol such as, for example, the polyethylene glycol / cholesterol conjugate, polyethylene glycol-phosphatidylethanolamine or better still polyethylene glycol / squalene.
• the polyethylene glycol / squalene conjugate is associated, in this case, with the nanoparticles according to the invention, and thus leads to the formation of coated nanoparticles. on the surface of polyethylene glycol.
• the polyethylene glycol / squalene conjugate advantageously acts during the process of forming the nanoparticles according to the invention as a surfactant because of its amphiphilic behavior and thus stabilizes the colloidal suspension, thus reducing the size of the nanoparticles formed. .
• the nanoparticles according to the invention are formulated in the form of an aqueous dispersion. According to another advantageous embodiment, the nanoparticles according to the invention are in the form of lyophilisate.
• these are 4,8- (3,4-dihydroxy-5-oxo-2) -4,8-dihydroxy-5-oxo-2, 4,8,13,17,21-pentamethyl-docosa- 4,8,12,16,20-pentaenoate nanoparticles.
• 5-Dihydro-furan-2-yl) -2-hydroxyethyl also referred to as 2- (3,4-dihydroxy-5-oxo-2,5-dihydro) -1,1 ', 2-trisnor-squalenate furan-2-yl) -2-hydroxy-ethyl or squalenoyl-ascorbic acid or squalenoyl-vitamin C
• 6-squalenoyl-ascorbic acid nanoparticles also called 6-squalenoyl-vitamin C.
• nanoparticles of 5- (1,2-dihydroxyethyl) -3-hydroxyl-4- ⁇ [(8E, 12E, 16E) -4, 8, 13, 17,21-pentamethyldocosa - 4,8,12,16,20-pentaen-1-yl] oxy ⁇ -2,5-dihydrofuran-2-one also named nanoparticles of 3-squalenyl-ascorbic acid or nanoparticles of 3 -squalenyl-vitamin vs.
• the nanoparticles are formed by placing the complex in the presence of an aqueous medium under conditions conducive to its agglomeration in the state of nanoparticles. It may in particular be methods called nanoprecipitation or emulsion / evaporation of solvent.
• nanoparticles according to the present invention may advantageously be obtained in the following manner.
• a complex or conjugate according to the invention is dispersed in at least one organic solvent (for example an alcohol such as ethanol or acetone) in a concentration sufficient to obtain, when the resulting mixture is added, with stirring, and generally drip, to an aqueous phase, the instantaneous formation of nanoparticles according to the invention in suspension in said aqueous phase.
• organic solvent for example an alcohol such as ethanol or acetone
• the nanoparticles are isolated according to the techniques well known to those skilled in the art. Nanoprecipitation can generally be performed at room temperature. Whatever it is, the temperature of implementation must not affect the activity of the vitamin C molecule considered.
• the process for preparing the nanoparticles according to the invention is particularly advantageous insofar as it does not necessarily require the presence of surfactants.
• Another advantage of the present invention is that no organic solvent or any potentially toxic surfactant is required for the preparation of said compositions according to the invention.
• surfactants generally advantageously devoid of any toxicity
• This type of surfactant can also provide access to even smaller sizes during the formation of nanoparticles.
• polyoxyethylene-polyoxypropylene copolymers there may be mentioned polyoxyethylene-polyoxypropylene copolymers, phospholipid derivatives and lipophilic derivatives of polyethylene glycol.
• polyethylene glycol cholesterol As the lipophilic derivative of polyethylene glycol, mention may be made, for example, of polyethylene glycol cholesterol.
• polyoxyethylene-polyoxypropylene block copolymers there may be mentioned polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymers, also called poloxamers ® , pluronics ® or synperonics and which are marketed, in particular, by the company BASF.
• poloxamines which consist of hydrophobic segments (based on polyoxypropylene), hydrophilic segments (based on polyoxyethylene) and a central part deriving from the ethylene diamine unit can also be used.
• a complex and / or nanoparticles that are suitable for the invention may advantageously be formulated in a composition that may be in any galenical form normally available for the chosen mode of administration.
• a composition may be in any galenical form normally available for the chosen mode of administration.
• the choice of the form considered for the complex to know particulate or nonparticulate can be made taking into account the nature of the medium of pharmaceutical formulation particularly considered.
• the complex in the case of a monophasic or multiphase formula such as an emulsion, may advantageously be formulated in the form of nanoparticles in the aqueous phase.
• a composition of the invention comprises a physiologically acceptable medium.
• physiologically acceptable medium is meant a non-toxic environment that can be applied to the skin and has a pleasant appearance, odor and feel.
• a composition of the invention may be a cosmetic, dermatological or pharmaceutical composition.
• composition of the invention may be administered topically.
• a composition of the invention administered topically can advantageously be formulated in any dosage form suitable for the care of the skin and mucous membranes and may be in the form of ointment, creams, milks, ointments, solutions, gels, sprays, lotions, or suspensions.
• a composition of the invention may also be in the form of a transdermal system allowing an active or passive release of the nanoparticles according to the invention by transdermy, for example patch or gel patch (hydrogel).
• transdermy for example patch or gel patch (hydrogel).
• compositions are prepared according to the usual methods.
• Formulation agents and excipients for topical composition are known in these fields and are not the subject of a detailed description herein.
• the compositions in accordance with the invention may contain other active ingredients which may be beneficial to take advantage of, together with the effect of vitamin C.
• the present invention relates to a food supplement comprising at least one complex according to the invention or nanoparticles according to the invention.
• oral compositions according to the invention can be carried out by any usual method known to those skilled in the art to produce oral solutions, dragees, capsules, gels, emulsions, swallowing tablets or chewable, capsules, especially soft or hard capsules, dissolving granules, syrups, solid or liquid foods and hydrogels.
• a complex according to the invention and / or nanoparticles according to the invention can also be incorporated as such in all forms of food supplements or fortified foods, for example food bars, milk drinks or not.
• Example 1a Preparation of 6-squalenoyl-vitamin C (VitC-SO),
• the solution is concentrated under reduced pressure to provide the acid chloride of crude 1,1 ', 2-tris-nor-squalenic acid as a yellow oil.
• a stream of dry HC1 hydrochloric acid is bubbled to saturation in 10 mL of N-methylpiperidone (NMP) placed in a Durand wash bottle (caked).
• NMP N-methylpiperidone
• NMP remaining in the residue is distilled under reduced pressure (0.05 mm Hg, 60 ° C.) to give a thick residue which is chromatographed on silica gel, eluting with ethyl acetate and then with a mixture of AcOEt / MeOH, 98: 2 to provide 6-squalenoyl-vitamin C as a pale yellow thick oil (190 mg, 34%).
• the mixture is stirred at -15 ° C for 30 min, then the reaction is allowed to warm to room temperature while stirring for a further 2 h.
• the reaction mixture is then concentrated, the residue is taken up in 20 ml of ethyl acetate and the organic phase is washed successively with a saturated solution of sodium bicarbonate (3 ml) and then with a saturated solution of NaCl (3 ml). .
• the organic phases are dried over magnesium sulphate and concentrated under reduced pressure.
• the crude mixture was purified by flash chromatography on silica gel eluting with cyclohexane / AcOEt (1: 1) to give fully protected squalenyl ascorbic acid as a colorless oil (438 mg, 67%).
• the crude product is purified by RP-18 silica gel column chromatography eluting with a mixture of acetonitrile / H 2 O (95: 5) to give 3-squalenyl-ascorbic acid (3-SQ Vit C). (293 mg, 37%) as a pale yellow oil.
• the nanoparticles are obtained by the precipitation / solvent evaporation method, by analogy with the method described in Fessi et al., Int. J. Pharm., 55, 1989, R1-R4.
• a solution of 7.6 mg of vitamin C-squalenized in ethanol (0.5 mL) is added dropwise to 1 mL of an aqueous solution (MilliQ ® water) containing 3% Pluronic ® F68 with magnetic stirring (500 rpm). Particles form instantly.
• the bottle containing the VitC-SQ solution is rinsed with 0.1 ml of ethanol and the rinsing solution is added to the suspension of nanoparticles.
• the suspension of nanoparticles is transferred into a calibrated 100 ml flask and concentrated under reduced pressure in a rotavapor (50-100 mbar at 20 ° C. for 10 min and then at 37 ° C. for approximately 3- 5 minutes) to a weight of 0.8 - 0.9 g.
• the solution is then supplemented to 1.0 g using MilliQ ® water.
• VitC-SQ nanoparticles are polydispersed.
• the method of precipitation / solvent evaporation provides nanoparticles of an average size of 51 nm with low polydispersity (0.08).
• the size of the nanoparticles obtained is measured with a nanosizer (Zetasizer ® from Malvern).
• a solution of 3-SQ Vit C (5.6 mg) in 0.5 ml of ethanol is added dropwise with stirring (500 rpm) to 1.5 ml of milliQ water. Precipitation of nanoparticles occurs spontaneously. Stirring is continued for 2 or 3 minutes.
• the nanoparticle suspension is then transferred to a calibrated round bottom flask and the ethanol is distilled under reduced pressure (50-100 mbar) for about 10 min at room temperature and then at 30 ° C for about 3-5 min. Evaporation is continued to a weight of the contents around 1.3 to 1.4 g. The weight of the suspension is then adjusted to 1.5 g with milliQ water or a 5% aqueous dextrose solution.
• the average hydrodynamic diameter of the nanoparticles was determined at 20 ° C by quasi-elastic light scattering at an angle of 90 ° with a Malvern Nanosizer (Malvern Instruments SA, Orsay, France) in a quartz cuvette (length 10 mm path). The samples were prepared by dilution 10 times in the water of the stock solution. Mean hydrodynamic diameter is given as a weighted number on all populations (mean Z). An average diameter of 69 nm with a polydispersity index of 0.22 was observed. The nanoparticles are stable over a period of three days.
• Explants of human skin with a diameter of about 10 nm, obtained from surgical plastic surgery waste and more particularly from a plasty abdominal women about thirty years old are used.
• the human skin is maintained in survival in a usual nutrient medium at 37 ° C in a humid atmosphere, enriched with 5% of C0 2 , for ten days.
• the explants of human skin are treated using a gel according to the invention comprising the complex or VitC-SQ conjugate according to the invention obtained, for example, formulated in squalene as described in Example 3 to obtain compositions comprising respectively 1% and 3% by weight, expressed by weight of vitamin C active relative to the total weight of the composition.
• a gel according to the invention comprising the complex or VitC-SQ conjugate according to the invention obtained, for example, formulated in squalene as described in Example 3 to obtain compositions comprising respectively 1% and 3% by weight, expressed by weight of vitamin C active relative to the total weight of the composition.
• concentration of the VitC-SQ complex is adjusted accordingly.
• the first control trial consisted of untreated human skin explants (control # 0).
• control No. 1 The other two control tests use, respectively, as a vitamin C derivative, "free” vitamin C (control No. 1) and a Vitamin C-palmitate derivative, also named hereafter VitC-palm (control No. 2) which can be provided by Sigma-Aldrich or Alpha Aesar.
• These two active ingredients are used at a concentration of 1% and 3% by weight, expressed as weight of vitamin C active, relative to the total weight of the composition.
• a gel comprising the VitC-Palm derivative formulated in squalene is prepared according to the method indicated in Example 3 in which VitC-SQ is replaced by VitC-Palm.
• compositions tested is applied topically, on the basis of
• the samples After 48 hours of fixation in the ordinary Bouin, the samples are dehydrated and impregnated with paraffin using a Leica 1020 dehydration automaton. Then, they are put in bulk according to the MO-H-153 operating procedure. using a Leica EG 1160 coating station.
• Sections of 5 ⁇ are made using a Minot microtome, Leica
• the epidermis has 4 to 5 cell bases with good morphology and the dermoepidermal junction has a clear relief.
• the papillary dermis shows thick collagen fibers forming a more or less dense and well-cellularized network.
• acid GAGs are not moderate in epidermal inter-cellular spaces as well as in the papillary dermis.
• VitC-SQ according to the invention at 1% slight epidermal stimulation
• VitC-SQ according to the invention at 3% net epidermal stimulation
• the microscopic observations are made by optical microscopy, using a Leica microscope type DMLB, at the objective x 40.
• the shots were taken with Olympus DP72 camera and Olympus Cell A D acquisition and archiving software.
• Collagen I The marking of Collagen I is carried out on frozen sections with an antibody anti-human collagen I rabbit (ref Monosan PS047, polyclonal), at l / 800th for 1 hour at room temperature with an amplifier system biotin / streptavidin, revealed in fluorescence (FITC caltag SA 1001). The nuclei were stained with propidium iodide.
• collagen III it is marked on in paraffin (formalin fixation) with polyclonal antibody anti-human collagen III goat (SBA ref: 1330-1301), at l / 50 th for overnight at room temperature using a Universal Vectastain RTU VECTOR avidin / biotin enhancer system, revealed in diaminobenzidine (DAB) with nuclei against stained with Masson hemalum.
• DAB diaminobenzidine
• VitC-SQ derivatives allow dermal and epidermal stimulation more important than VitC-Palm.
• the complex according to the invention has an anti-aging activity superior to the commercial derivative VitC-Palm.
• test products are added to the wells in different concentrations. After 4 hours of incubation, the culture medium is replaced by new biological medium and the cells were incubated for 48 hours at 37 ° C. under 5% CO 2 .
• the culture medium is removed by inversion and 100 PBS / well with MTT at a concentration of 0.5 mg / ml are added.
• the plate is returned to the incubator for 2 hours at 37 ° C under 5% CO 2 .
• 100 ⁇ l, of a solution of SDS (sodium dodecyl sulphate) are added to each well and then the plate is re-incubated for 12 h at 37 ° C. under 5% of C0 2 .
• the spectrophotometric measurements are performed on an ELISA plate reader at the wavelength of 570 nm.
• the percentages of viability of the cells in the presence of the test molecules are then calculated by means of the following formula: (average OD of the treated cells - average control OD alone / average control cell OD (100% viability) - middle control OD only) x 100 ⁇ standard deviation; or presented in the form of curves tracing the evolution of the absorbance as a function of time.
• the IC50 (50% inhibitory concentration” is the concentration inhibiting the growth of 50% of the cells) then correspond to the concentration of the tested product causing a loss of 50% in cell viability (see Table below).
• VitC-SQ is half as toxic as VitC-palm.

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